• Sizing of YAC Clones

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Donis-Keller Lab Manual, Department of Genetics in Washington University School of Medicine http://hg.wustl.edu/hdk_lab_manual/yeast/yeast8.html

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Purpose:

To determine the size of a yeast artificial chromosome within the background of the normal yeast chromosomal complement.

Time required:

5 days

Specialized Equipment:

• BioRad CHEF DR-II pulsed field gel electrophoresis box

Specialized Reagents:

• Yeast Lysis Buffer

• Lambda ladder DNA / AB1380 standard plugs

• Lyticase (Sigma #L-8137)

Procedure:

Day 1-2

1. Inoculate a single colony of the yeast clone of interest into 5-7 ml of AHC medium in a 15 ml snap cap tube. Place tube on roller drum in 30 degrees C incubator for 2-3 days (optimal cultures have a reddish hue usually seen after 2 days).

Day 3

1. Centrifuge at 2500 rpm for 10 minutes in Beckman J-6 centrifuge; pour off supernatant and resuspend cell pellet in 10 ml of 50 mM EDTA (pH 8.0). Centrifuge at 2500 rpm for 10 minutes, pour off supernatant, and repeat this wash once more.

2. Resuspend washed cell pellet in 100 祃 of SCE by gentle mixing; hold tubes at room temperature for 3-5 minutes.

3. Add 100 祃 of SCE containing 70 mM Dithiothreitol (DTT) and 280 units/ml of Lyticase. Incubate at 37 degrees C for 2 hours with occasional gentle mixing every 15 minutes to produce spheroplasts.

4. Mix 280 祃 of 1% low melt 0.5X TBE agarose (in 125 mM EDTA) with each sample, and mix by pipeting up and down with a P-1000 pipetman. Pipette 200-250 祃 into individual plug molds that accompany the CHEF DR apparatus (there should be enough for 2-3 plugs). Allow to cool to room temperature for 10-15 minutes, then place on ice (or refrigerate) for 15 minutes.

5. Remove plugs and place in a Corning 6 well plate; add 4-6 ml of freshly made Yeast Lysis buffer to each plug containing well (several plugs from one clone can be placed in each well). Incubate at 50 degrees C with gentle rocking for 2 hours to overnight. Place a layer of parafilm between the wells and plate cover to prevent evaporation. Plugs will lose their reddish tinge and become an opaque white color during this step.

Day 4

1. Place plugs in 50 mM EDTA with 2.5 units/ml of RNase at 4 degrees C for long term storage (these will remain stable for about 1-2 months). Plugs to be run out on a CHEF gel are equilibrated at room temperature in 0.5X TBE with gentle agitation, over 2 hours with 2-3 changes of buffer.

2. The temperature of the 0.5X TBE buffer needs to be maintained between 12-14 degrees C during electrophoresis; turn on the chiller 3 hours ahead of time, then add 2 liters of 0.5X TBE and allow buffer to circulate for 1 hour before the start of each run to achieve proper running temperatures.

3. Pour a 1% agarose gel (0.5X TBE) into the BioRad CHEF gel mold. When the agarose has set, place a 5 mm slice of each plug into the wells; standard lanes should contain specially constructed lambda ladder and plugs from the yeast strain AB1380. Pipette 1% TBE agarose (approximately 250 祃) over each plug to fill the well. Place the gel in the BioRad CHEF DR apparatus, and run with a ramped switching time of 20 seconds to 100 seconds for 24 hours, at 200 volts (some prefer to use 60 seconds switching time for 15 hours, followed by 9 hours at 90 seconds).

Day 5

1. At the end of the run, place the gel in 1 liter of dH2O containing 20 祃 of 10 mg/ml ethidium bromide and gently rock for 30 minutes. Destaining by placing gel in ddH2O and rocking for 15-30 minutes may be helpful at this point to increase the sharpness of bands, but is not absolutely necessary. Photograph the gel as per routine. Yeast artificial chromosomes will appear as an extra band relative to the normal yeast complement of chromosomes. The size can be determined by comparing to the AB1380 standards lane, or to the lambda ladder.

2. If Southern transfer is desired, first soak the gel in 0.25N HCl for 30 minutes to depurinate the large DNA fragments. Rinse briefly in ddH2O, then denature the DNA by gently rocking in 1.5M NaCl, 0.5N NaOH for 30 minutes. Rinse briefly in ddH2O, then place on a stack of blot blocks with one sheet of 3MM Whatman paper. Carefully remove all the bubbles by rolling with a test tube, then place nylon membrane over it. Place another layer of Whatman over this, followed by 6 blot blocks, paper towels, and a weight. Use 1.5M NaCl, 0.25N NaOH as transfer buffer. Allow to transfer overnight, with frequent changes of buffer. Wash and bake the membranes as per routine Southern transfer procedure.
   

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